Irdye 680

We used 12% sodium dodecyl sulfate (SDS) polyacrylamide gels to separate 5 µg of total protein from hTau40- and pcDNA3.2-transfected SH-SY5Y differentiated cells before transferring them onto a PVDF (polyvinylidene fluoride) membrane. A tau protein ladder (rPeptide, Watkinsville, GA USA) containing all 6 human isoforms of tau and a recombinant tau 441 (hTau40) protein (rPeptide) were also loaded on the gel as controls. PVDF membranes were blocked for 1 h using Intercept^® Blocking Buffer TBS (LI-COR Biosciences) and probed overnight at 4 °C with anti-phospho-Tau (Threonine 181) and anti-total Tau (Tau 12) antibodies at a 1:1000 dilution overnight at 4 °C. Membranes were then incubated with secondary antibody (either IRDye^® 680 or IRDye^® 800CW) at a 1:10,000 dilution for 1 h at room temperature and scanned using the Odyssey Infrared Imaging System (LI-COR Biosciences).

Cytokeratins or vimentin were immobilized on 96-wells microtiter plates (200 ng in 50 μl of PBS) (4°C, overnight), blocked with Odyssey blocking buffer for 1h at 37°C and incubated with Tc85-11^LamG recombinant protein in PBS (60 μg/ml). Wells were then washed 3 times with PBS and bound protein detected using an anti-gp85 monoclonal antibody (G1G8, kindly provided by Dr. Eliciane Mattos), followed by anti-mouse immunoglobulin-Ig antibodies conjugated with IRDye680 (LI-COR Biosciences). Plates were then analyzed with the Odyssey Imaging System (LI-COR Biosciences). The monoclonal antibody G1G8 recognizes a neutralizing epitope (sequence TGETPLEPFGFCFGA) within the LamG domain of the Tc85-11 protein [14 (link)]. For the competition experiments, the Tc85-11^LamG recombinant protein in PBS (60 μg/ml) was incubated in the absence or presence of peptides (200 μM).

U-Omp19 samples were run under both non-reducing and reducing conditions by SDS-PAGE as described previously [18] (link). For WB, after being subjected to SDS-PAGE, samples were transferred onto a nitrocellulose membrane. A polyclonal rabbit anti-U-Omp19 serum followed by anti-rabbit IgG labeled with IRDye 680 (LI-COR Biosciences) were used to visualize the bands. Images were captured and documented with an Odyssey infrared image-scanner (LI-COR Biosciences).

Western blot analysis of experimental and control cell lysates was carried out using the Odyssey^® Infrared System (LI-COR Biosciences, Lincoln, NE) and polyclonal anti-phospho-AMPK-α (Thr 172) antibody (Cat # 07-681SP), polyclonal Anti-p21 antibody (Cat # 0506550), and polyclonal Anti-Kip1 (p27) (Cat # 060445) were purchased from Millipore (Millipore Corporation, Iselin, NJ ). Polyclonal beta actin antibody (Cat # sc-1615) and polyclonal RASSF1C antibody (sc-18724) were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA), and fluorescently-labeled secondary antibodies IRDye^® 680 and 780 RD Infrared Dye were purchased from LI-COR (LI-COR Biosciences, Lincoln, NE). The experiments were repeated at least 3 times. Protein levels were normalized to actin levels (the loading control).

APP-CTM1, APP-CT11, PS1_NT, Flotilin-2 and APP_NTH-452 homemade rabbit polyclonal antibodies were generously provided by Dr. Gopal Thinakaran (University of Chicago, Chicago, IL) as described previously (Deyts et al., 2012 (link); Vetrivel et al., 2009 (link)). Monoclonal anti-MAP2, GAP-43 (clone 7B10) and GAPDH were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal DCC (clone G97-449) antibody was purchased from BD Biosciences (San Diego, CA). Polyclonal phospho-(Ser/Thr) PKA substrate antibody and phospho-CREB (Ser133) antibodies were purchased from Cell Signaling Technology (Danvers, MA) and EMD Millipore (Billerica, MA), respectively. Monoclonal Alexa-647 and Alexa-555, and polyclonal Alexa-555 secondary antibodies were purchased from Invitrogen (Carlsbad, CA). IRDye 680 and IRDye 800CW-conjugated secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). γ-secretase inhibitor Compound E was generously provided by Dr. Todd E. Golde (University of Florida, Gainesville, FL) (Seiffert et al., 2000 (link)). Cis-N-(2-phenylcyclopentyl) azacyclotridec-1-en-2-amine (MDL,12-330A) was obtained from Enzo Life Science (Farmingdale, NY). Tetrodotoxin was purchased from Tocris Bioscience (distributed by Fisher Scientific, Pittsurgh, PA). Unless indicated, all other reagents were purchased from Sigma-Aldrich.